The present invention relates to a conjugate vaccine for Neisseria meningitidis comprising detoxified lipooligosaccharide from which at least one primary O-linked esterified fatty acid has been removed from lipid A linked to an immunogenic carrier. More specifically, the invention relates to a conjugate vaccine against N. meningitidis in which the detoxified lipooligosaccharide does not contain the structure of the lacto-N-neotetraose human blood group antigen.
Neisseria meningitidis is a capsulated gram-negative bacterium that causes meningitis and septic shock in humans. In industrialized countries, the annual incidence is 1 to 5 in 100,000, while in nonindustrialized countries, it is estimated that 330,000 persons suffer from meningococcal disease, with 35,000 deaths per year. More than half of the cases occur in children below the age of 5 years, with the highest incidence occurring in the first 2 years. About 90% of the cases are caused by serogroups A, B, and C; the remainder are caused by serogroups Y and W135. Group A dominates in Africa during both epidemic and endemic periods, while group B is the most medically relevant in the United States and Europe.
The sole natural habitat and reservoir for N. meningitidis is the human upper respiratory mucosal surface, primarily the nasopharynx. Meningococci are transmitted by large respiratory droplets or direct contact with respiratory secretions. Carriage of the meningococcus in general does not lead to disease. During non-epidemic periods, when disease is rare, 5-30% of the adult population is colonized by the meningococcus which suggests that host rather than bacterial factors determine the outcome.
There are two major problems in the development of vaccines for N. meningitidis. First, the current A, C, Y, and W135 capsular polysaccharide (PS) vaccines are less immunogenic in young children, especially infants; and second, group B capsular PS, a polymer of xcex1(2-8)-linked sialic acid which is also present in some human gangliosides and a number of fetal glycoproteins, is poorly immunogenic in humans. The immunogenicity of the capsular PSs can be improved by coupling them to proteins, but this procedure may not be desirable for group B capsular PS because antibodies to the PS may cross-react with human tissue antigens and may cause an autoimmune disease. There is no vaccine against group B meningococcal strains which are the most important disease strains in the United States and Europe.
Lipooligosaccharide (LOS) is a major N. meningitidis cell surface antigen. LOS contains both lipid A and oligosaccharide (OS) components. LOS from N. meningitidis has twelve immunotypes, L1-L12. Immunotypes L8, L9, L10 and L11 are found within group A meningococci, of which L9, L10 and L11 are prevalent and uniquely associated with this serogroup. Immunotypes L1 to L8 are identified within groups B and C meningococci. Because the lipid A component of LOS is toxic, it is detoxified prior to conjugation to an immunogenic carrier for use in a vaccine. Munford et al. (U.S. Pat. No. 5,103,661) describes removal of secondary O-linked esterified fatty acids from bacterial lipopolysaccharide (LPS) from various Gram negative bacteria, including N. meningitidis, by treatment with the enzyme acyloxyhydrolase. This treatment removes two of the four O-linked fatty acid chains from lipid A. The resulting xe2x80x9cdetoxifiedxe2x80x9d lipid A is only 30-60 fold less toxic than native lipid A (Erwin et al., Infect. Immun. 59:1881-1887, 1991), which corresponds to a toxicity of 167-333 IU/xcexcg which is unacceptable for vaccine use as stated in the W.H.O. Technical Report Series 814:15-37, 1991 (p. 22, section A.3.3.6).
Hydrazine has been used to detoxify lipid A from various bacterial species, including nontypeable Haemophilus influenzae (Gu et al., Infect. Immun. 64:4047-4053, 1996), Shigella (polotsky et al., Infect. Immun. 62:210-214, 1994) and Moraxella catarrhalis (Gu et al., Infect. Immun. 66:1891-1897, 1998). The resulting detoxified LOS or LPS (dLOS or DeALPS) were conjugated to carrier proteins and their immunogenicities were determined. The hydrazine-detoxified dLOS conjugates from Nontypeable H. influenzae and M. catarrhalis were immunogenic. However, the hydrazine-detoxified LPS from Shigella was poorly immunogenic. Thus, it cannot be predicted whether an LOS or LPS detoxified by hydrazine treatment will be immunogenic.
Thus, there is a need for an effective vaccine against N. meningitidis. The present invention addresses this need.
One embodiment of the present invention is a conjugate vaccine for Neisseria meningitidis, comprising N. meningitidis lipooligosaccharide which does not contain a lacto-N-neotetraose (LNnT) antigen from which at least one primary O-linked fatty acid has been removed (dLOS), and an immunogenic carrier covalently linked thereto. The conjugate vaccine may also be multivalent, composed of dLOSs from different strains and/or immunotypes of N. meningitidis. In another aspect of the present invention, the immunogenic carrier is a protein. Preferably, the protein is tetanus toxin/toxoid, CRM 197, outer membrane proteins from gram negative bacterial pathogens such as high molecular weight proteins, P6 and P4 from nontypeable Haemophilus influenzae; CD and USPA from Moraxella catarrhalis, diphtheria toxin/toxoid, detoxified P. aeruginosa toxin A, cholera toxin/toxoid, pertussis toxin/toxoid, Clostridium perfringens exotoxins/toxoid, hepatitis B surface antigen, hepatitis B core antigen, rotavirus VP 7 protein, or respiratory syncytial virus F and G protein. In one aspect of this preferred embodiment, the protein is tetanus toxoid. Advantageously, both primary and secondary O-linked fatty acids have been removed from the lipooligosaccharide.
Another embodiment of the invention is a conjugate vaccine against Group B N. meningitidis, comprising at least one, and preferably more than one, LOS(s) isolated from non-Group B immunotypes of N. meningitidis, the LOS(s) not containing lacto-N-neotetraose, the LOS(s) detoxified by removing at least one primary O-linked fatty acid, and the detoxified LOS(s) (dLOS) raising antibodies which cross-react with LOS(s) from Group B N. meningitidis strains.
The present invention also provides a conjugate vaccine for N. meningitidis, comprising N. meningitidis lipooligosaccharide which does not contain a LNnT antigen from which at least one O-linked fatty acid has been removed (dLOS), and an immunogenic carrier covalently linked thereto via a linker. Preferably, the linker is adipic acid dihydrazide, xcex5-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine or p-nitrophenylamine; most preferably, the linker is adipic acid dihydrazide.
Another embodiment of the invention is isolated N. meningitidis lipooligosaccharide detoxified by removal of at least one O-linked fatty acid therefrom. Preferably, the detoxified lipooligosaccharide is at least about 5,000 fold less toxic than the lipooligosaccharide as determined using the limulus amebocyte lysate (LAL) assay. In another preferred embodiment, the detoxified lipooligosaccharide is at least about 10,000 fold less toxic than the lipooligosaccharide as determined using the limulus amebocyte lysate (LAL) assay. Preferably, the lipooligosaccharide is detoxified by removal of both primary and secondary O-linked fatty acids.
The present invention also provides a pharmaceutical composition comprising the vaccine conjugates described above in a pharmaceutically acceptable carrier. The pharmaceutical composition may further comprise an adjuvant. Preferably, the adjuvant is alum or monophosphoryl lipid A.
Another embodiment of the invention is a method of producing antibodies which recognize N. meningitidis, comprising administering to an individual an effective antibody-producing amount of the vaccine described above. The route of administration may be intramuscular, subcutaneous, intraperitoneal, intraarterial, intravenous or intranasal; most preferably, the administering step is intramuscular. According to another aspect of this preferred embodiment, the effective dose is between about 10 xcexcg and about 50 xcexcg. The method may further comprise booster injections of between about 10 xcexcg and about 25 xcexcg.
According to another aspect of the invention, there is provided a method of detoxifying lipooligosaccharide from N. meningitidis, comprising removing at least one primary O-linked fatty acid therefrom. Preferably, the ester-linked fatty acid(s) is removed by treating the LOS with hydrazine.
Still another aspect of the present invention is a method of making a conjugate vaccine against N. meningitidis, comprising: removing at least one primary O-linked fatty acid from N. meningitidis lipooligosaccharide which does not contain a LNnT antigen to produce dLOS; and covalently binding the dLOS to an immunogenic carrier. Advantageously, the removing step comprises treatment with hydrazine. The method may further comprise the step of attaching dLOS to a linker and attaching the linker to the carrier. Preferably, the linker is adipic acid dihydrazide, xcex5-aminohexanoic acid, chlorohexanol dimethyl acetal, D-glucuronolactone, cystamine or p-nitrophenylethyl amine; most preferably, the linker is adipic acid dihydrazide. In one aspect of this preferred embodiment, the dLOS is at least about 5,000 fold less toxic than the lipooligosaccharide as determined using the limulus amebocyte lysate (LAL) assay. In another aspect of this preferred embodiment, the dLOS is at least about 10,000 fold less toxic than the lipooligosaccharide as determined using the LAL assay.